1. Field of the Invention
The present invention relates generally to synthetic ganglioside derivatives, not found in nature, which are useful as immunosuppressive agents. More particularly, the present invention relates to glycosphingolipids, artificial anchor gangliosides and simplified carbohydrate moiety-gangliosides which are useful pharmaceutical agents for inhibiting an immune response.
2. Description of Related Art
Although the immune response is often seen as beneficial, in certain circumstances the immune response to an antigen can actually be harmful to the animal in which the immune response occurs. An example where the immune response creates a condition wherein the host is subject to serious pathologic sequelae is in such autoimmune diseases as lupus erythematosus, rheumatoid arthritis, diabetes, and Crohn's disease. In autoimmune diseases, the immune response is directed against host tissues, and therefore use of immunosuppressive agents is a treatment approach.
Another, and one of the most important, areas which often requires substantial immunosuppression is tissue transplantation, where the suppression of the immune response is crucial in order to prevent graft rejection by the host (host versus graft reaction, HVG) and graft rejection of the host (graft versus host rejection, GVH). Typically, the tissue which is grafted is allogeneic, where the inhibition of alloreactive T lymphocytes by immunosuppressive agents is essential to the prevention of allograft rejection. Depending upon the nature of the allograft (i.e. liver, kidney, or bone marrow), the course of immunosuppressive therapy may be relatively brief (months) or may have to be continued indefinitely (years to lifetime). All of the immunosuppressive agents used thus far have significant drawbacks relating either to direct toxicity on other organ systems or to failure to provide "balanced" immunosuppression. The latter problem has two distinct aspects; on one hand inadequate suppression of the immune response can lead to rejection, while on the other hand excessive immunosuppression can allow the development of opportunistic infections and neoplasia. Thus, the need to develop an effective nontoxic immunosuppressive agent which does not cause the above severe complications continues.
At present, multi-drug therapy, including cytotoxic agents, is utilized following organ transplantation. This typically comprises combination therapy, such as treatment with cyclosporin A, azathioprine, and prednisone, the rationale being that each drug acts at a different stage in the immune response and the combination therapy will require lower doses of each individual drug, thus diminishing their dose-related side effects. However, the side effects remain significant while the efficacy of this form of therapy is still not satisfactory. Rejection continues to account for nearly 50% of graft losses in renal transplantation. And, distinguishing rejection from cyclosporin A nephrotoxicity may be difficult.
Another major cause of graft loss is systemic infection, usually by opportunistic infections, which require the tapering or cessation of immunosuppression, which leads to graft loss. Also, with such combination therapy in transplantation, there has been a significant increase in the incidence of lymphomas (Wilkinson, et al., "Transplantation," 47:293-296, 1989). The chronic failure of immunosuppressive therapy is revealed by the fact that the graft survival rate of 85% at 1 year drops to 67% at 5 years (Kahan, et al., "Am J. Kidney Dis," 5:288-295, 1985) in recipients of cadaveric renal transplants receiving triple therapy. Clearly, then the existing immunosuppressive therapy is inadequate. This has stimulated the search for, and development of, new immunosuppressive drugs, and particularly agents that are not directly toxic to either the immune system or to other organ systems. One approach to overcoming the problems associated with present immunosuppressive drugs is the use of biological agents which are actually produced by the animal. An example of such biological agents are the gangliosides.
Gangliosides are a class of glycosphingolipids. As shown schematically in FIG. 1, gangliosides have a structure containing a carbohydrate moiety linked to a ceramide. The carbohydrate moiety includes a sugar moiety which has at least one monosaccharide and one or more sialic acid moiety(s), i.e. sialic acid groups (N-acetyl or N-glycolyl neuraminic acid). FIG. 2 sets forth the nomenclature which is used to describe the ceramide moiety. The ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion. The number to the left of the colon indicates the carbon chain length of the fatty acid or long chain base, and the number to the right indicates the degree of unsaturation. The major long chain base structures (to the left of the dash) of normal human brain gangliosides are d18:1 and d20:1, and of extraneural gangliosides, d18:1. The major fatty acid structures (to the right of the dash) are 18:0 and 20:0.
Gangliosides are also classified according to the number of monosaccharides in the carbohydrate moiety and the number of sialic acid groups present in the sialic acid moiety(s); Further classification is dependent upon where and how many sialic acid(s) are bound to the carbohydrate moiety. For example, the international symbol G.sub.M1a designates one of the more common gangliosides which has been extensively studied. The subscript, "M" in the symbol indicates that the ganglioside is a monosialoganglioside and "1" indicates that there are four saccharide units present in the carbohydrate moiety. The subscripts "a", "b" or "c" indicate isomers of the particular ganglioside described which differ in the position of the sialic acid(s). The subscripts "D", "T" and "Q" used as international ganglioside symbols represents gangliosides, trisialongangliosides and tetrasialongangliosides, respectively. The subscripts "2", "3" and "4" represent trisaccharide, disaccharide and monosaccharide gangliosides, respectively. The terminal saccharide is the saccharide which is located at the end of the carbohydrate moiety which is opposite to the end that is attached to the ceramide moiety.
Ten common human brain gangliosides and their biosynthetic pathway are set forth in the FIG. 3. The structure of each ganglioside is set forth using conventional abbreviations for the ceramide, saccharide and sialic acid (SA) groups. FIG. 3 also outlines the biosynthetic pathway of the gangliosides. The biosynthesis of gangliosides is discussed in detail in S. Roseman, Chem. Phys. Lipids, 5: 270-297, 1970.
It is well know that gangliosides are functionally important in the nervous system and it has been claimed that gangliosides are useful in the therapy of peripheral nervous system disorders. Numerous gangliosides are derivatives thereof have been used to treat a wide variety of nervous system disorders including cerebral ischemic strokes. For example, see U.S. Pat. Nos. 4,940,694; 4,937,232; and 4,716,223. Gangliosides have also been used to affect the activity of phagocytes (U.S. Pat. No. 4,831,021) and to treat gastrointestinal disease-producing organisms (U.S. Pat. No. 4,762,822).
The use of gangliosides and ganglioside analogues to suppress or to otherwise affect the immune system has not yet been investigated as extensively as their use in neurological disorders.
The first report of ganglioside suppression of immune responses in vivo was published twenty years ago by Agarwal and Neter, who discovered inhibition by gangliosides of the primary antibody response to bacterial antigens in mice (Agarwal, et al., J. Immunol.,107: 1448-1456, 1971). Recent studies have shown that tumor gangliosides which are shed in vivo enhance tumor formation in mice (Ladisch, et al., J.Clin.Invest., 79:1879-1882, 1987), a finding confirmed by other laboratories (Allessandri, et al., Cancer Res. 47:4243-4347, 1987; Saha, et al., Int.J.Cancer,41:432-435, 1988); indirect evidence (Ladisch, et al., J. Clin. invest., 79:1879-1882, 1987) suggests that this enhancement occurs by an immunologic mechanism. However, a recent investigation into the in vivo immunosuppressive effect of G.sub.M1 ganglioside or mixed bovine brain gangliosides (mainly G.sub.M1), G.sub.D1a, G.sub.D1b, and G.sub.T1b) was conducted by Presti, D. et al., (Presti, D. et al. J. Neuroimmunology, 22: 233-239, 1989). The study concluded that there was no evidence of a suppressive effect on humoral or cellular immunity exhibited in vivo by the G.sub.M1 ganglioside or the mixed brain gangliosides.
As noted above, gangliosides are composed of three elements. The role these elements play, however, in the immunosuppressive activity of gangliosides is unknown. Indeed, in the past, the identification of preferred active ganglioside structures has largely been limited to naturally occurring gangliosides. Although naturally occurring gangliosides vary to some extent in the structure of their elements, the available variants do not permit a full exploration of the role the various elements play in immunosuppression.
There is, thus, a continuing need to develop chemically synthesized gangliosides, wherein the various elements of naturally occurring gangliosides are replaced with synthetic or artificial moieties.